Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades.

ETHNOPHARMACOLOGICAL RELEVANCE: Cortex fraxini (also known as Qinpi), the bark of Fraxinus rhynchophylla Hance and Fraxinus stylosa Lingelsh, constitutes a crucial component in several traditional Chinese formulas (e.g., Baitouweng Tang, Jinxiao Formula, etc.) and has demonstrated efficacy in alleviating intestinal carbuncle and managing diarrhea. Cortex fraxini has demonstrated commendable anticancer activity in the realm of Chinese ethnopharmacology; nevertheless, the underlying mechanisms against colorectal cancer (CRC) remain elusive. AIM OF THE STUDY: Esculin, an essential bioactive compound derived from cortex fraxini, has recently garnered attention for its ability to impede viability and induce apoptosis in cancer cells. This investigation aims to assess the therapeutic potential of esculin in treating CRC and elucidate the underlying mechanisms. MATERIALS AND METHODS: The impact of esculin on CRC cell viability was assessed using CCK-8 assay, Annexin V/PI staining, and Western blotting. Various cell death inhibitors, along with DCFH-DA, ELISA, biochemical analysis, and Western blotting, were employed to delineate the modes through which esculin induces HCT116 cells death. Inhibitors and siRNA knockdown were utilized to analyze the signaling pathways influenced by esculin. Additionally, an azomethane/dextran sulfate sodium (AOM/DSS)-induced in vivo CRC mouse model was employed to validate esculin's potential in inhibiting tumorigenesis and to elucidate its underlying mechanisms. RESULTS: Esculin significantly suppressed the viability of various CRC cell lines, particularly HCT116 cells. Investigation with diverse cell death inhibitors revealed that esculin-induced cell death was associated with both apoptosis and ferroptosis. Furthermore, esculin treatment triggered cellular lipid peroxidation, as evidenced by elevated levels of malondialdehyde (MDA) and decreased levels of glutathione (GSH), indicative of its propensity to induce ferroptosis in HCT116 cells. Enhanced protein levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and p-eIF2α suggested that esculin induced cellular endoplasmic reticulum (ER) stress, subsequently activating the Nrf2/ARE signaling pathway and initiating the transcriptional expression of heme oxygenase (HO)-1. Esculin-induced excessive expression of HO-1 could potentially lead to iron overload in HCT116 cells. Knockdown of Ho-1 significantly attenuated esculin-induced ferroptosis, underscoring HO-1 as a critical mediator of esculin-induced ferroptosis in HCT116 cells. Furthermore, utilizing an AOM/DSS-induced colorectal cancer mouse model, we validated that esculin potentially inhibits the onset and progression of colon cancer by inducing apoptosis and ferroptosis in vivo. CONCLUSIONS: These findings provide comprehensive insights into the dual induction of apoptosis and ferroptosis in HCT116 cells by esculin. The activation of the PERK signaling pathway, along with modulation of downstream eIF2α/CHOP and Nrf2/HO-1 cascades, underscores the mechanistic basis supporting the clinical application of esculin on CRC treatment.

Exosomal miR-1470 is a diagnostic biomarker and promotes cell proliferation and metastasis in colorectal cancer.

BACKGROUND: In recent years,the lack of specific markers for the diagnosis of colorectal cancer has led to an upward trend in both morbidity and mortality from this condition. There is an urgent need to identify molecular biomarkers that contribute to early cancer detection. This study aimed to identify specific exosomal microRNAs that hold potential as diagnostic biomarkers for CRC. METHODS: We screened for differentially expressed miRNAs using the CRC exosome dataset GSE39833. To validate the results in the public database, we collected serum from 168 CRC patients and 168 healthy volunteers. The expression levels of exosomal miR-1470 in healthy volunteers and CRC patients were analyzed using qRT-PCR. To evaluate the diagnostic potential of the selected miR-1470 in distinguishing CRC patients from healthy controls, we analyzed its receiver operating characteristic curve. To explore the biological functions of miR-1470 in CRC cell lines, we detected the miR-1470's ability to regulate the growth and metastasis of CRC cells by CCK8, transwell and other assays after transfection of miR-1470 in SW480, HCT-116 cells. RESULTS: Exosomal miR-1470 exhibited significant up-regulation in CRC patients compared to healthy volunteers. The ROC curve analysis revealed an area under the curve (AUC) of 0.74 (95% confidence interval: 0.6876-0.7920) for exosomal miR-1470, indicating its potential as a diagnostic biomarker. Furthermore, the expression level of miR-1470 in CRC patients showed correlations with age, metastasis, and HDL content. We overexpressed miR-1470 in CRC cell lines. CCK8 proliferation assay showed that miR-1470 promoted the proliferation ability of SW480 and HCT-116 cells. Transwell assay showed that miR-1470 promoted the migration and invasion ability of SW480 and HCT-116 cells. CONCLUSION: This suggested that non-invasive diagnosis of CRC is possible by detecting the level of miR-1470 in exosomes, which has important implications for early detection and treatment of this disease.

Increased PKN2 and M2-Polarized Macrophages Promote HCT116 Cell Invasion.

Colorectal cancer is the third most common malignant tumor, with highly invasive and metastatic potential in the later stage. This study investigated the role of PKN2 overexpression and M2-polarized macrophages in dictating the malignant phenotype of colorectal cancer cells. HCT116 colorectal cancer cell line with PKN2 overexpression was generated to investigate the functional role of PKN2. THP-1 cells were polarized into M2-like macrophages, and the co-culture system of THP-1/M2 cells and HCT116 cells was established to examine the impacts of M2-polairzed macrophages on the malignant phenotype of colorectal cancer cells. PKN2 overexpression promoted cell proliferation, migration and invasion in HCT116 colorectal cancer cells, and reduced spontaneous cell death in the cell culture. Besides, the presence of M2-polarized THP-1 cells significantly enhanced the aggressive phenotype of HCT116 cells. Both PKN2 overexpression and M2-polarized THP-1 cells increased the expression of NF-κB p65 in HCT116 cells, indicating that enhanced NF-κB signaling may contribute to the augmented aggressiveness of HCT116 cells. These findings suggest PKN2 as an oncogenic factor in colorectal cancer and that M2-polarized THP-1 cells may promote the progression of colorectal cancer by activating NF-κB signaling.

Melittin as an Activator of the Autophagy and Unfolded Protein Response Pathways in Colorectal HCT116 Cell Line.

Background: The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the UPR and autophagy as two critical pathways involved in tumorigenesis of colorectal and drug resistance. This study aimed to investigate the effect of melittin on these pathways in the colorectal cancer (CRC) HCT116 cells. Methods: MTT method was carried out to assess the cytotoxicity of melittin on the HCT116 cell line for 24, 48, and 72 h. After selecting the optimal concentrations and treatment times, the gene expression of autophagy flux markers (LC3-ßII and P62) and UPR markers (CHOP and XBP-1s) were determined using qRT-PCR. The protein level of autophagy initiation marker (Beclin1) was also determined by Western blotting. Results: MTT assay showed a cytotoxic effect of melittin on the HCT116 cells. The increase in LC3-ßII and decrease in P62 mRNA expression levels, along with the elevation in the Beclin1 protein level, indicated the stimulatory role of melittin on the autophagy. Melittin also significantly enhanced the CHOP and XBP-1s expressions at mRNA level, suggesting the positive role of the melittin on the UPR activation. Conclusion: This study shows that UPR and autophagy can potentially be considered as two key signaling pathways in tumorigenesis, which can be targeted by the BV melittin in the HCT116 cells. Further in vivo evaluations are recommended to verify the obtained results.

Angelica gigas polysaccharide induces CR3-mediated macrophage activation and the cytotoxicity of natural killer cells against HCT-116 cells via NF-κB and MAPK signaling pathways.

Angelica gigas (A. gigas) is traditional medicinal herb that mainly exists in Korea and northeastern China. There have been relatively few studies conducted thus far on its polysaccharides and their bioactivities. We purified and described a novel water-soluble polysaccharide derived from A. gigas and investigated its immunoenhancing properties. The basic components of crude and purified polysaccharides (F1 and F2) were total sugar (41.07% - 70.55%), protein (1.12-10.33%), sulfate (2.9-5.5%), and uronic acids (0.5-31.05%) in total content. Our results demonstrated that the crude and fractions' molecular weights (Mw) varied from 42.2 to 285.2 × 103 g/mol. As the most effective polysaccharide, F2 significantly stimulated RAW264.7 cells to release nitric oxide (NO) and express several cytokines. Furthermore, F2 increased the expression of tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-É£), natural killer cytotoxicity receptors (NKp44), and granzyme-B in NK-92 cells and enhanced the cytotoxicity against HCT-116 cells. In our experiments, we found that F2 stimulated RAW264.7 cells and NK-92 cells via MAPK and NF-κB pathways. The monosaccharide and methylation analysis of the high immunostimulant F2 polysaccharide findings revealed that the polysaccharide was primarily composed of 1 â†’ 4, 1 â†’ 6, 1 â†’ 3, 6, 1 â†’ 3 and 1 â†’ 3, 4, 6 galactopyranose residues, 1 â†’ 3 arabinofuranose residues, 1 â†’ 4 glucopyranose residues. These results demonstrated that the F2 polysaccharide of A. gigas which possesses potential immunostimulatory attributes, could be used to create a novel functional food.

Real-time simultaneous refractive index and thickness mapping of sub-cellular biology at the diffraction limit.

Mapping the cellular refractive index (RI) is a central task for research involving the composition of microorganisms and the development of models providing automated medical screenings with accuracy beyond 95%. These models require significantly enhancing the state-of-the-art RI mapping capabilities to provide large amounts of accurate RI data at high throughput. Here, we present a machine-learning-based technique that obtains a biological specimen's real-time RI and thickness maps from a single image acquired with a conventional color camera. This technology leverages a suitably engineered nanostructured membrane that stretches a biological analyte over its surface and absorbs transmitted light, generating complex reflection spectra from each sample point. The technique does not need pre-existing sample knowledge. It achieves 10-4 RI sensitivity and sub-nanometer thickness resolution on diffraction-limited spatial areas. We illustrate practical application by performing sub-cellular segmentation of HCT-116 colorectal cancer cells, obtaining complete three-dimensional reconstruction of the cellular regions with a characteristic length of 30 µm. These results can facilitate the development of real-time label-free technologies for biomedical studies on microscopic multicellular dynamics.

Discovery of a stilbenoid-flavanone hybrid as an antitumor Wnt/ß-catenin signaling pathway inhibitor.

A series of designed stilbenoid-flavanone hybrids featuring sp3-hybridized C2 and C3 atoms of C-ring was evaluated against colorectal cancers presented compounds 4, 17, and 20 as the most potential compounds among explored compounds. Evaluation of the anticancer activity spectrum of compounds 4, 17, and 20 against diverse solid tumors presented compounds 17 and 20 with interesting anticancer spectrum. The potencies of compounds 17 and 20 were assessed in comparison with FDA-approved anticancer drugs. Compound 17 was the, in general, the most potent showing low micromolar GI50 values that were more potent than the standard FDA-approved drugs against several solid tumors including colon, brain, skin, renal, prostate and breast tumors. Compound 17 was subjected for evaluation against normal cell lines and was subjected to a mechanism study in HCT116 colon cancer cells which presented it as an inhibitor of Wnt signaling pathway triggering G2/M cell cycle arrest though activation of p53-p21 pathway as well as intrinsic and extrinsic apoptotic death of colon cancer cells. Compound 17 might be a candidate for further development against diverse solid tumors including colon cancer.

Unravelling a novel CTNND1-RAB6A fusion transcript: Implications in colon cancer cell migration.

The interchange of DNA sequences between genes may occur because of chromosomal rearrangements leading to the formation of chimeric genes. These chimeric genes have been linked to various cancers, accumulated significant interest in recent times. We used paired-end RNA-seq. data of four CRC and one normal sample generated from our previous study. The STAR-Fusion pipeline was utilized to identify the fusion genes unique to CRC. The in-silico identified fusion gene(s) were explored for their diagnostic, prognostic and therapeutic biomarker potential using TCGA-datasets, then validated through PCR and DNA sequencing. Further, cell line-based studies were performed to gain functional insights of the novel fusion transcript CTNND1-RAB6A, which was amplified in one sample. Sequencing revealed that there was a total loss of the CTNND1 gene, whereas RAB6A retained its coding sequence. Further, RAB6A was functionally characterized for its oncogenic potential in HCT116 cell line. RAB6A under-expression was found to be significantly associated with increased cell migration and is proposed to be regulated via the RAB6A-ECR1-Liprin-α axis. We conclude that RAB6A gene may play significant role in CRC oncogenesis, and could be used as a potential biomarker and therapeutic target in future for better management of a subset of CRCs harbouring this fusion.

Selective Synergy of Rapamycin Combined With Methioninase on Cancer Cells Compared to Normal Cells.

BACKGROUND/AIM: Rapamycin and recombinant methioninase (rMETase) have both shown efficacy to target cancer cells. Rapamycin prevents cancer-cell growth by inhibition of the mTOR protein kinase. rMETase, by degrading methionine, targets the methionine addiction of cancer and has been shown to improve the efficacy of chemotherapy drugs. In the present study, we aimed to determine if a synergy exists between rapamycin and rMETase when used in combination against a colorectal-carcinoma cell line, compared to normal fibroblasts, in vitro. MATERIALS AND METHODS: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line and Hs-27 human fibroblasts were determined using the CCK-8 Cell Viability Assay. After calculating the IC50 of each drug, we determined the efficacy of rapamycin and rMETase combined on both HCT-116 and Hs-27. RESULTS: Hs-27 normal fibroblasts were more sensitive to rapamycin than HCT-116 colon-cancer cells (IC50=0.37 nM and IC50=1.38 nM, respectively). HCT-116 cells were more sensitive to rMETase than Hs-27 cells (IC50 0.39 U/ml and IC50 0.96 U/ml, respectively). The treatment of Hs-27 cells with the combination of rapamycin (IC50=0.37 nM) and rMETase (IC50=0.96 U/ml) showed no significant difference in their effect on Hs-27 cell viability compared to the two drugs being used separately. However, the treatment of HCT-116 cells with the combination of rapamycin (IC50=1.38 nM) and rMETase (IC50=0.39 U/ml) was able to decrease cancer-cell viability significantly more than either single-drug treatment. CONCLUSION: Rapamycin and rMETase, when used in combination against colorectal-cancer cells, but not normal fibroblasts, in vitro, have a cancer-specific synergistic effect, suggesting that the combination of these drugs can be used as an effective, targeted cancer therapy.

GNAS mutation inhibits growth and induces phosphodiesterase 4D expression in colorectal cancer cell lines.

Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.

Ethanolic extract from leaves of tithonia diversifolia induces apoptosis in HCT-116 cells through oxidative stress.

Tithonia diversifolia is a perennial bushy plant found in South America with significant ethnopharmacological importance as an antimalarial, antidiabetic, antibacterial, and anticancer agent. The aim of the present study was to determine the cytotoxicity of the ethanolic extract from leaves of T. diversifolia (TdE) on human cancer cell lines (HCT-116, SNB-19, NCIH-460 and MCF-7), as well as the mechanism of action involved in cell death and cellular modulation of oxidative stress. The TdE exhibited significant activity with IC50 values ranging from 7.12 to 38.41 µg/ml, with HCT-116 being the most sensitive cell line. Subsequent experiments were conducted with HCT-116 cell line. TdE decreased the number of viable cells, followed by induction of apoptotic events, increase in mitochondrial membrane permeabilization, and enhanced G2/M phase of the cell cycle. Pro-oxidative effects including elevated acidic vesicular organelle formation, lipid peroxidation, and nitric oxide by-products, as well as reduced levels of intracellular glutathione and reactive oxygen species production were also observed following incubation with TdE, which may lead to DNA damage followed by apoptotic cell death. These results demonstrate the potential of TdE ethanolic leaf extraction for biological activity and enhance the importance of continuing to study natural sources of plants for the development of anticancer agents.

Methylation of NRIP3 Is a Synthetic Lethal Marker for Combined PI3K and ATR/ATM Inhibitors in Colorectal Cancer.

INTRODUCTION: The aim of this study was to investigate the epigenetic regulation and underlying mechanism of NRIP3 in colorectal cancer (CRC). METHODS: Eight cell lines (SW480, SW620, DKO, LOVO, HT29, HCT116, DLD1, and RKO), 187 resected margin samples from colorectal cancer tissue, 146 cases with colorectal adenomatous polyps, and 308 colorectal cancer samples were used. Methylation-specific PCR, Western blotting, RNA interference assay, and a xenograft mouse model were used. RESULTS: NRIP3 exhibited methylation in 2.7% (5/187) of resected margin samples from colorectal cancer tissue, 32.2% (47/146) of colorectal adenomatous polyps, and 50.6% (156/308) of CRC samples, and the expression of NRIP3 was regulated by promoter region methylation. The methylation of NRIP3 was found to be significantly associated with late onset (at age 50 years or older), poor tumor differentiation, lymph node metastasis, and poor 5-year overall survival in CRC (all P < 0.05). In addition, NRIP3 methylation was an independent poor prognostic marker ( P < 0.05). NRIP3 inhibited cell proliferation, colony formation, invasion, and migration, while induced G1/S arrest. NRIP3 suppressed CRC growth by inhibiting PI3K-AKT signaling both in vitro and in vivo . Methylation of NRIP3 sensitized CRC cells to combined PI3K and ATR/ATM inhibitors. DISCUSSION: NRIP3 was frequently methylated in both colorectal adenomatous polyps and CRC. The methylation of NRIP3 may potentially serve as an early detection, late-onset, and poor prognostic marker in CRC. NRIP3 is a potential tumor suppressor. NRIP3 methylation is a potential synthetic lethal marker for combined PI3K and ATR/ATM inhibitors.

Exploring the apoptotic effects of sericin on HCT116 cells through comprehensive nanostring transcriptomics and proteomics analysis.

Sericin, a silk protein from Bombyx mori (silkworms), has many applications, including cosmetics, anti-inflammation, and anti-cancer. Sericin complexes with nanoparticles have shown promise for breast cancer cell lines. Apoptosis, a programmed cell death mechanism, stops cancer cell growth. This study found that Sericin urea extract significantly affected HCT116 cell viability (IC50 = 42.00 ± 0.002 µg/mL) and caused apoptosis in over 80% of treated cells. S-FTIR analysis showed significant changes in Sericin-treated cells' macromolecule composition, particularly in the lipid and nucleic acid areas, indicating major cellular modifications. A transcriptomics study found upregulation of the apoptotic signaling genes FASLG, TNFSF10, CASP3, CASP7, CASP8, and CASP10. Early apoptotic proteins also showed that BAD, AKT, CASP9, p53, and CASP8 were significantly upregulated. A proteomics study illuminated Sericin-treated cells' altered protein patterns. Our results show that Sericin activated the extrinsic apoptosis pathway via the caspase cascade (CASP8/10 and CASP3/7) and the death receptor pathway, involving TNFSF10 or FASLG, in HCT116 cells. Upregulation of p53 increases CASP8, which activates CASP3 and causes HCT116 cell death. This multi-omics study illuminates the molecular mechanisms of Sericin-induced apoptosis, sheds light on its potential cancer treatment applications, and helps us understand the complex relationship between silk-derived proteins and cellular processes.

Ku80 is indispensable for repairing DNA double-strand breaks at highly methylated sites in human HCT116 cells.

DNA double-strand breaks (DSBs) are harmful to mammalian cells and a few of them can cause cell death. Accumulating DSBs in these cells to analyze their genomic distribution and their potential impact on chromatin structure is difficult. In this study, we used CRISPR to generate Ku80-/- human cells and arrested the cells in G1 phase to accumulate DSBs before conducting END-seq and Nanopore analysis. Our analysis revealed that DNA with high methylation level accumulates DSB hotspots in Ku80-/- human cells. Furthermore, we identified chromosome structural variants (SVs) using Nanopore sequencing and observed a higher number of SVs in Ku80-/- human cells. Based on our findings, we suggest that the high efficiency of Ku80 knockout in human HCT116 cells makes it a promising model for characterizing SVs in the context of 3D chromatin structure and studying the alternative-end joining (Alt-EJ) DSB repair pathway.

Berberine and RNAi-Targeting Telomerase Reverse Transcriptase (TERT) and/or Telomerase RNA Component (TERC) Caused Oxidation in Colorectal Cancer Cell Line, HCT 116: An Integrative Approach using Molecular and Metabolomic Studies.

Colorectal cancer (CRC) is the most common cancer in both men and women and is associated with increased telomerase levels and activity. The potential downstream effects of TERT and/or TERC downregulation by berberine (a telomerase inhibitor) or RNA interference (RNAi) on various target RNAs, proteins, relative telomerase activity (RTA), relative telomere length (RTL), hydrogen peroxide concentration [H2O2], percentage of cell cycle distribution, cell size and granularity as well as cellular metabolites were explored in HCT 116 cell line. Knockdown of TERT decreased TERC. The downregulation of TERT and/or TERC caused increment of [H2O2], G0/G1 phase arrest in addition to decreased S and G2/M phases, as well as diminished cell size. RTL was later reduced as a result of TERT, TERT and/or TERC downregulation which decreased RTA. It was discovered that xanthine oxidase (XO) was significantly and positively correlated at FDR-adjusted p value < 0.05 with RTA, TERT, TERT, TERC, and RTL. HCT 116 with decreased RTA was closely clustered in the Principal Component Analysis (PCA) indicating similarity of the metabolic profile. A total of 55 metabolites were putatively annotated in this study, potentially associated with RTA levels. The Debiased Sparse Partial Correlation (DSPC) Network revealed that RTA was directly correlated to TERT. There were 4 metabolic pathways significantly affected by low level of RTA which include (1) purine metabolism, (2) glycine, serine, and threonine metabolism, (3) glyoxylate and dicarboxylate metabolism, and (4) aminoacyl-tRNA biosynthesis. The Gene-Metabolite Interaction Network implied that reduced RTA level was related to the mechanism of oxidative stress. This study reveals the linkages between RTA to various selected RNAs, proteins, metabolites, oxidative stress mechanism and subsequently phenotypic changes in HCT 116 which is valuable to understand the intricate biological interactions and mechanism of telomerase in CRC.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect.

BACKGROUND: Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects. METHODS: Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells. RESULTS: S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence. CONCLUSION: These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.

Apoptotic and antiproliferative effects of Inula viscosa L. water extract in the expression of microRnas on HCT 116 cell line: an in vitro study.

In this study, the antiproliferative and apoptotic effects of Inula viscosa L. water extract (IVE) on HCT 116 has been examined, and the change in the expression of miRNAs. Phenolic compounds of IVE were determined as µg/g extract using by HPLC-DAD. Quantitative determination of apoptosis, cell viability, IC50 values and miRNAs of the cells were determined during 24, and 48 hours. IVE contain coumarin, rosmarinic acid and chlorogenic acid. According to the findings of our study, the expression of miR-21 and miR-135a1 was upregulated, and miR-145 was downregulated in HCT 116 cells (Control). Additionally, IVE was found to have significant potential in regulating miRNAs, downregulating miR-21, miR-31 and miR-135a1, and upregulating miR-145 in HCT-116 cells. All these results show that the anticancer effect of IVE via regulating miRNAs' expression has been demonstrated for the first time, and may be candidate biomarkers in colorectal cancer.

A large-scale microRNA transcriptome-wide association study identifies two susceptibility microRNAs, miR-1307-5p and miR-192-3p, for colorectal cancer risk.

Transcriptome-wide association studies (TWAS) have identified many putative susceptibility genes for colorectal cancer (CRC) risk. However, susceptibility miRNAs, critical dysregulators of gene expression, remain unexplored. We genotyped DNA samples from 313 CRC East Asian patients and performed small RNA sequencing in their normal colon tissues distant from tumors to build genetic models for predicting miRNA expression. We applied these models and data from genome-wide association studies (GWAS) including 23 942 cases and 217 267 controls of East Asian ancestry to investigate associations of predicted miRNA expression with CRC risk. Perturbation experiments separately by promoting and inhibiting miRNAs expressions and further in vitro assays in both SW480 and HCT116 cells were conducted. At a Bonferroni-corrected threshold of P < 4.5 × 10-4, we identified two putative susceptibility miRNAs, miR-1307-5p and miR-192-3p, located in regions more than 500 kb away from any GWAS-identified risk variants in CRC. We observed that a high predicted expression of miR-1307-5p was associated with increased CRC risk, while a low predicted expression of miR-192-3p was associated with increased CRC risk. Our experimental results further provide strong evidence of their susceptible roles by showing that miR-1307-5p and miR-192-3p play a regulatory role, respectively, in promoting and inhibiting CRC cell proliferation, migration, and invasion, which was consistently observed in both SW480 and HCT116 cells. Our study provides additional insights into the biological mechanisms underlying CRC development.

ZNF70 regulates IL-1ß secretion of macrophages to promote the proliferation of HCT116 cells via activation of NLRP3 inflammasome and STAT3 pathway in colitis-associated colorectal cancer.

Chronic inflammation is a key driver for colitis-associated colorectal cancer (CAC). It has been reported that inflammatory cytokines, such as IL-1ß, could promote CAC. Zinc finger protein 70 (ZNF70) is involved in multiple biological processes. Here, we identified a previously unknown role for ZNF70 regulates macrophages IL-1ß secretion to promote HCT116 proliferation in CAC, and investigated its underlying mechanism. We showed ZNF70 is much higher expressed in CAC tumor tissues compared with adjacent normal tissues in clinical CAC samples. Further experiments showed ZNF70 promoted macrophages IL-1ß secretion and HCT116 proliferation. In LPS/ATP-stimulated THP-1 cells, we found ZNF70 activated NLRP3 inflammasome, resulting in robust IL-1ß secretion. Interestingly, we discovered the ZnF domain of ZNF70 could interact with NLRP3 and decrease the K48-linked ubiquitination of NLRP3. Moreover, ZNF70 could activate STAT3, thereby promoting IL-1ß synthesis. Noteworthy, ZNF70 enhanced proliferation by upregulating STAT3 activation in HCT116 cells cultured in the conditioned medium of THP-1 macrophages treated with LPS/ATP. Finally, the vivo observations were confirmed using AAV-mediated ZNF70 knockdown, which improved colitis-associated colorectal cancer in the AOM/DSS model. The correlation between ZNF70 expression and overall survival/IL-1ß expression in colorectal cancer was verified by TCGA database. Taken together, ZNF70 regulates macrophages IL-1ß secretion to promote the HCT116 cells proliferation via activation of NLRP3 inflammasome and STAT3 pathway, suggesting that ZNF70 may be a promising preventive target for treating in CAC.

Driver gene KRAS aggravates cancer-associated stroke outcomes.

The incidence of cancer-associated stroke has increased with the prolonged survival times of cancer patients. Recent genetic studies have led to progress in cancer therapeutics, but relationships between oncogenic mutations and stroke remain elusive. Here, we focused on the driver gene KRAS, which is the predominant RAS isoform mutated in multiple cancer types, in cancer associated stroke study. KRASG13D/- and parental human colorectal carcinoma HCT116 cells were inoculated into mice that were then subjected to a photochemically-induced thrombosis model to establish ischemic stroke. We found that cancer inoculation exacerbated neurological deficits after stroke. Moreover, mice inoculated with KRASG13D/- cells showed worse neurological deficits after stroke compared with mice inoculated with parental cells. Stroke promoted tumor growth, and the KRASG13D/- allele enhanced this growth. Brain RNA sequencing analysis and serum ELISA showed that chemokines and cytokines mediating pro-inflammatory responses were upregulated in mice inoculated with KRASG13D/- cells compared with those inoculated with parental cells. STAT3 phosphorylation was promoted following ischemic stroke in the KRASG13D/- group compared with in the parental group, and STAT3 inhibition significantly ameliorated stroke outcomes by mitigating microglia/macrophage polarization. Finally, we compared the prognosis and mortality of colorectal cancer patients with or without stroke onset between 1 January 2007 and 31 December 2020 using a hospital-based cancer registry and found that colorectal cancer patients with stroke onset within 3 months after cancer diagnosis had a worse prognosis. Our work suggests an interplay between KRAS and ischemic stroke that may offer insight into future treatments for cancer-associated stroke.